Keith Shelton - US grants

The clinical effects of lead intoxication are well known, but there is increasing concern over the possible effects of low level lead exposure. This proposal is directed at a nuclear protein which is a target for ingested lead. The protein, p32/6.3, is an abundant component of lead- induced intranuclear inclusion bodies in kidney. In normal adults it is most abundant in central nervous system neurons, the increase occurring as synapses mature. The long term objectives are to understand lead's effects on p32/6.3 in brain as well as in kidney and other cells where it is normally a low-abundance component, and further, to understand the consequences of these effects. A monoclonal antibody will be used to assay p32/6.3 pool size in several lead exposed models: cultured brain microvessel endothelial cells; cerebrum, cerebellum, brain microvessels, and kidney of adults and of neonates whose mothers are lead exposed; and kidney of adults after EDTA chelation therapy. Using information from a partial peptide sequence, the polymerase chain reaction and cDNA cloning will be used to complete the sequence; this may confirm homology with a cAMP-binding protein. Using cDNA sequence, fusion proteins will be used as antigens; the resultant antibodies will be used to perform immunocytochemistry in the models described above. A related project will study the mechanism of induction of a lead-induced protein.

DESCRIPTION: (Applicant's Abstract) Sedative/anxiolytic misuse and abuse are serious problems in our society. The most common of these drugs, benzodiazepine and barbiturates, are positive allosteric modulators of GABA-A receptor function. GABA-A receptors are heteropentameric assemblies from at least five subunit families (alpha 1-6, beta 1-4, gamma 1-3, delta and epsilon). In vitro studies have shown that GABA-A receptor subunit composition is critical to the actions of GABA ligands. The presence of a delta subunit in recombinant GABA-A receptors confers increased sensitivity to GABA and insensitivity to benzodiazepines and neurosteroids. Recently, mice lacking a functional GABA-A delta subunit have been created. Neurosteroid-induced sleep time is decreased in these animals, in direct contrast to predictions based on recombinant receptors. This suggests that multiple compensatory changes in subunit composition may occur as a result of delta subunit deletion. This application will use the drug discrimination paradigm to examine the receptor-mediated subjective discriminative stimulus effects of diazepam in delta subunit knockout mice. The discriminative stimulus effects of a drug are a function of specific receptor interactions and are believed to be related to abuse liability. The first specific aim of this proposal is to establish a diazepam discrimination in delta knockout mice and wild-type controls. It is hypothesized that both strains of mice will successfully acquire the discrimination but will differ in the time required to reach criteria performance. The second major aim is to determine if the delta subunit plays a role in the discriminative stimulus effects of diazepam and other GABA-A modulators. Based on data showing altered neurosteroid effects in delta knockout mice, we believe that each strain will exhibit a unique substitution profile of GABA-A modulators for diazepam. These data will provide an important characterization of the discriminative stimulus effects of diazepam in delta subunit knockouts and would be the first study of the discriminative stimulus effects of a drug in any knockout mouse. Additionally, the results from this study may help to elucidate how changes in specific subunit alterations of the GABA receptor translate into changes in the behavioral effects of sedative/anxiolytic drugs.

Alcohol abuse and alcoholism are serious societal problems. Genetics play a major role in determining the risk for developing alcohol abuse disorders. Inbred and transgenic mice are powerful tools for examining the genetic and molecular basis of the effects of alcohol. Some transgenic and inbred mice have altered responses to the physiological and reinforcing effects of ethanol. It has not been determined if the subjective discriminative effects of ethanol are different across strains of inbred mice or altered in transgenic mice. We propose to examine the discriminative stimulus properties of ethanol in two inbred mouse strains (C57BL/6 and DBA/2J) and one transgenic mouse line which over-expresses 5-HT3 receptors. The C57BL/6 mouse strain is characterized by high levels of ethanol drinking and decreased sensitivity to ethanol. The DBA/2J and 5-HT3 over-expressing strains drink less ethanol than controls and have increased responses to the motor activating effects of low dose ethanol. The first specific aim of this proposal is to train an ethanol discrimination in 5-HT3 over- expressers, B6SJL control mice, C57BL/6 mice and DBA/2J mice. It is hypothesized that all four strains will be capable of discriminating ethanol from vehicle but will differ in sensitivity to ethanol's discriminative stimulus effects. The second specific aim is to determine if test drug substitution patterns for ethanol systematically differ between transgenic and inbred mouse strains. It is hypothesized that genetic alterations of the 5-HT3 receptor system will effect not only the ability of 5-HT3 ligands to substitute for ethanol, but also drugs which act via the GABAA and NMDA receptor systems. It is also hypothesized that C57BL/6 and DBA/2J mice will differ from one another in the substitution patterns of the test drugs for ethanol. These studies will characterize the discriminative stimulus effects of ethanol in transgenic and inbred strains of mice. These experiments will also explore the direct consequences of alterations of the 5-HT3 receptor systems to the discriminative stimulus effects of ethanol anal the contribution of genetic. background to the ethanol's discriminative ctimnlnc effects.

DESCRIPTION (provided by applicant): Inhalant abuse presents a serious, under-appreciated public health problem in the United States and worldwide. Basic research on inhalants has lagged far behind that on most other classes of abused drugs. Several factors may be responsible for the paucity of scientific studies in the area. Lack of appropriate methods to explore the abuse-related in vivo neurochemical effects of inhalants is clearly one factor. However, another more fundamental impediment to advancing our basic understanding of inhalants is the sheer size and heterogeneity of the class itself. It is currently unclear to what extent inhalants should even be considered a formal drug class since it is the only class based upon route of administration rather than pharmacological mechanisms of action. It is our hypothesis that inhalant drug discrimination in mice can be used to gain basic information concerning the in vivo abuse-related neurochemical effects of inhalants as well as permit inhalants to be grouped into pharmacologically-based subclasses. In Aim 1 we will train groups of mice to discriminate one of four different inhalants from air: toluene, 1,1,1-trichlroethane (TCE), isoflurane or nitrous oxide. We will subsequently conduct cross-tests among these four compounds as well as with other representative volatile solvents and volatile anesthetics. We hypothesize that these cross-tests will reveal several distinct inhalant groups. If confirmed these findings would support our contention that subclasses of inhalants can be differentiated based on their discriminative stimuli. In Aim 2 we will determine the pharmacological mechanisms of action of toluene, TCE, isoflurane and nitrous oxide. In the case of toluene and TCE we will focus on exploring the GABAA receptor positive modulatory effects of these compounds revealed in our prior studies. Specifically we will perform cross-tests and antagonism experiments with compounds that selectively modulate GABAA receptors composed of particular subunits. In the case of the isoflurane discrimination we will also examine other potential targets based on our preliminary data as well as the available in vivo and in vitro literature. As we lack preliminary data with nitrous oxide our choice of cross-test compounds will initially be based on the literature and then further refined as sufficient drug discrimination results are generated. In Aim 3 will we confirm the pharmacological mechanisms of action underlying our theoretical inhalant subclasses. We will train animals to discriminate three additional inhalant selected based on their similarity to toluene, TCE and isoflurane and conduct cross-tests with compounds identified in Aim 2 as being capable of differentiating between these hypothetical inhalants subclasses. A similar pattern of cross-substitution results with these newly trained inhalants will confirm the pharmacological validity of our inhalant classification framework as well as provide important insights on the neuropharmacology of these compounds.

In 2015 over half a million Americans ages 12 and older were regular inhalant users. Inhalant use can result in profound neurological and organ damage and use is correlated with mental health treatment, delinquent behaviors and the abuse of other drugs. Despite the scope of the problem inhalants are the most poorly understood and chronically understudied of all the major classes of abused drugs, a problem exacerbated by a lack of resources, relevant technical expertise and validated methods. While our understanding of the effects of a few highly abused inhalants such as toluene has slowly advanced despite the difficulties, emerging inhalant threats have been almost completely unexplored. This is especially true with regard to the abuse of chlorocarbon gases. ?Dusting? as abuse of chlorocarbon gas products is referred, has contributed to a large number of well-publicized deaths due directly to the product effects or as a result of reckless behaviors while under the influence of the product. Alarmingly, chlorocarbon abuse may be supplanting abuse of other inhalants in the U.S., especially in adults, perhaps due to the perception of decreased risk or the fact that unlike other inhalants with strong odors, dusters have no lingering indications of use. The overarching goal of this R21 Developmental/Exploratory grant is to determine the extent to which two of the most commonly available chlorocarbons 1,1,1,2-tetrafluoroethane (R134a) and chloroethane engender abuse-related behaviors. We will accomplish this goal through two aims conducted in mice. Aim 1 will use male and female mice to assess the effects of each chlorocarbon on acute locomotor activity as well as if repeated administration induces locomotor sensitization. Sensitization is a phenomenon whereby the initial effect of a drug is enhanced following repeated administration and a characteristic of many drugs of abuse. We believe the demonstration of sensitization resulting from chlorocarbon exposure would provide a strong rationale for more detailed studies on the effects of these compounds on abuse-related neurocircuitry. Aim 2 will examine if chlorocarbons will facilitate intracranial self-stimulation behavior, demonstrating the extent to which chlorocarbon gases positively modulate brain reward mechanisms. Further, by comparing ICSS facilitation produced by chlorocarbons to that of toluene, arguably the most frequently abused inhalant, we believe it will be possible to ascertain the relative abuse liability of these chemicals. To maximize the potential to make predictions and the translational value of our data we will use both male and female subjects and have designed out studies to explore the impact of each of the chlorocarbon gases across estrous cycle phase. Taken as a whole this exploratory/developmental analysis will provide the critical foundation necessary for more detailed experiments aimed at understanding the neurochemistry underlying the abuse-related effects of chlorocarbon dusters.

Use of E-cigarette (ECs) devices has increased dramatically over recent years. Newly developed high-output ECs have the potential to delivery a variety of highly addictive drugs in addition to nicotine. Since inhalation produces nearly instantaneous CNS access it raises the alarming possibility that both opioid and psychomotor stimulant abuse by vaping may become serious public health concerns. However, at this time little is known about the reinforcing effects of drugs, other than nicotine, delivered using ECs and appropriate animal models are lacking. The present proposal seeks to fill this vacuum using a newly developed rodent vapor-puff delivery self-administration system to assess the reinforcing effects of fentanyl (FEN) and methamphetamine (METH) vapor. We will utilize the well-established fading principle to engender self-administration of FEN or METH vapor in separate groups of male and female rats. Rats will initially be trained in daily 1-hr operant sessions to emit lever responses in order to activate a nose poke device for delivery of a sweetened milk solution. The dipper cup delivering the milk will be elevated into the vapor delivery aperture thereby bringing the rat's nose into contact with vapor puffs. Over successive daily training sessions, 3 sec exposures to increasing concentrations of FEN or METH in e-juice vapor (50% vegetable glycerol/49% propylene glycol/1% yellow cake flavor) will occur concurrently with milk reinforcer presentation. Once behavior for the combination of the liquid reinforcer and vaped FEN or METH has reached stable, behaviorally-active levels we will discontinue the paired administration of milk reinforcers through a gradual daily fading procedure until responding is maintained by only FEN or METH vapor delivery. After vapor self-administration is established we will install an additional vaporizer activated by the lever-presses on a second lever permitting the use of a choice paradigm to examine the reinforcing effects of a range of FEN or METH concentrations. During dose-effect curve testing, responses on one lever of the operant chamber, signaled by a solidly illuminated lever-light, will occasion the opportunity to engage in nose-pokes that deliver drug-laden vapor whereas nose pokes following a response on the opposite lever, signaled by a blinking lever-light, will deliver drug-free vapor acting as a control that is identical in all respects aside from the presence of drug. As a second complimentary method of assessing the reinforcing effects of FEN or METH vapor we will utilize a progressive ratio test procedure. In addition to varying drug concentration we will parametrically explore the role of vaporizer output wattage in modulating the reinforcing effects of drug vapor. Lastly, commercial e-juice is composed of flavors, vegetable glycerol (VG) and propylene glycol (PG). The addition of flavor may play an important role in promoting and maintaining self-administration. To test this hypothesis we will systematically vary e-juice concentrations of flavor additive to examine the extent to which it impacts the reinforcing effects of FEN or METH vapor.